Nitric oxide was chosen as the "Molecule of the Year" by Science in 1992. The work supported by this grant, "Hepatocyte:Kupffer Cell Interactions in Surgical Sepsis," was cited in partial support of that choice. We were instrumental in the discovery of the high-output inducible nitric oxide synthase (iNOS) enzyme in parenchymal cells, hepatocytes, Kupffer cells, chondrocytes, cardiac myocytes, and smooth muscle cells. We showed that iNOS gene activation in each cell type was differentially regulated by synergistic stimuli, frequently a mixture of cytokines and microbial products. That hepatocyte iNOS expression was important in vivo as well as in vitro, in humans as well as in rodents, and that iNOS expression was of functional importance in these cells which produced it, were impacted by this grant. In addition, both our accumulated data and the data of numerous other laboratories have served to expand our understanding of the importance of iNOS in almost every aspect of the inflammatory response. Although the field has grown rapidly, much is still unknown about iNOS regulation and function. In recent years, we have focused our attention on the regulation of iNOS in hepatocytes. To this end, we have cloned the cDNA and the gene for human iNOS (the first human iNOS cloned) and began characterizing the transcriptional and post-transcriptional mechanisms regulating the expression and function of iNOS. In addition, we have studied the importance of substrate and cofactors in hepatocyte iNOS regulation in vivo and in vitro. We plan to continue this single-minded focus on hepatocyte iNOS regulation in two aims. AIM I is to define the role of hepatocyte phenotype, matrix proteins, and growth factors in the regulation of iNOS expression. In this aim, we will seek the mechanisms by which stresses that alter hepatocyte phenotype (such as the acute phase response or heat shock response) alter iNOS regulation. In addition, we will analyze the mechanisms by which various growth and differentiation hepatocyte phenotypes alter their expression of iNOS and how iNOS expression changes the hepatocyte phenotype. In AIM II, we will define the regulatory factors which govern the post-translational expression of iNOS enzyme, including post-translational modifications of the protein plus substrate and cofactor availability. We have recently obtained iNOS knockout mice and stable transformed human liver cell lines; we have recently developed systems of permanently and transiently transfecting human iNOS into these cells. Such techniques now permit an independent analysis of the effects of cytokines on post-translational iNOS modifications of structure and function. Only by understanding the regulation of the expression of this critical enzyme, activated by stress and sepsis, can important therapeutic interventions be designed.